The objective of the proposed project is to elucidate the molecular mechanism of the gene action in humans through analysis of the protein and genomic structures of normal and variant enzymes. The inherited deficiency of the X-linked glucose-6-phosphate dehydrogenase (G6PD) and the X-lined phosphoglycerate kinase (PGK) are associated with various clinical problems, and these two enzymes will be subjected to examination. In the proposed project, the genomic structure of G6PD, including the 5'-promoter region, will be determined by restriction mapping and nucleotide sequencing. The NH2-terminal structure of a putative nascent G6PD will be elucidated by primer-extension analysis of mRNA. Extensive exploration of the restriction fragment-length polymorphisms of the G6PD locus, and detailed mapping of the "G6PD cluster" locus (i.e., genes for hemophilia A- factor VIII, colorblindness, adrenoleukodystrophy, and fragile X- mental retardation) will be undertaken. The exact molecular lesion of the G6PD variants (i.e., associated with chronic and/or drug- or food-induced hemolytic anemia, those associated with functional abnormalities, the common variants existing in various populations, an over-production variant, and a "null" variant) and that of the PGK variants, associated with severe enzyme deficiency, chronic hemolytic anemia and mental disorders, will be determined by analysis of their genomic structures and by mass spectrometric analysis of their protein structures. In addition to the X-linked G6PD, a G6PD-like locus exists on chromosome 17. This locus will be cloned by screening a human genomic library in the cosmid vector. It will then be examined to determine whether the locus is for a non-functional pseudogene or for a functional gene encoding another G6PD isozyme or other enzyme. The three-dimensional structures of horse and yeast PGK are detailed. The structural/functional relationships of the normal and variant human PGK will be visualized by computer graphics analysis. These studies will extend our knowledge of the genomic structures, gene expressions, and genetic abnormalities of the G6PD and PGK loci. The studies may also provide us with valuable information about the clinically important "G6PD cluster" locus.